From olive leaves to spherical nanoparticles by one-step RESS process precipitation

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https://doi.org/10.1007/s00217-022-04127-3 ORIGINAL PAPER

From olive leaves to spherical nanoparticles by one‑step RESS process precipitation

A. Montes¹ · E. Merino¹ · D. Valor¹ · M. C. Guamán‑Balcázar² · C. Pereyra¹ · E. J. Martínez de la Ossa¹

Received: 20 July 2022 / Revised: 14 September 2022 / Accepted: 17 September 2022

Abstract

In this work, spherical nanoparticles to be used in cosmetic, agro food or pharmaceutical industries have been directly precipitated from olives leaves in one-step RESS process. The leaves were brought into contact with supercritical CO₂, and a fraction of the compounds from the flavone and flavonol families that can be found in the leaves were dissolved; then, by depressurizing the vessel, these compounds formed particles in the nanometer range. A complete factorial design was generated to thoroughly determine the influence from the main parameters on the RESS process with respect to the precipitated nanoparticles and their heterogeneity. Their antioxidant activity was also evaluated. Different pressures (250–350 bar), temperatures (60 and 100 °C), leaves sample weights (2 and 4 g) and cosolvent volumes, namely ethanol (9 and 18 mL), were studied as the main parameters that could affect the solvation and precipitation of the particle with active compounds in the leaves. Other parameters such as contact time (1 h) or nozzle size diameter (100 μm) remained unchanged. The antioxidant activity was evaluated by means of the radical scavenging method using the radical 2,2-diphenyl-1-picrylhydrazole (DPPH).

Spherical particles with diameters in the range of 55 nm to 4 µm were obtained. Lower pressures and higher temperatures seemed to result in a reduction of the mean particle size. Greater volume of cosolvent is also recommended to reduce mean particle size. However, lower pressure, temperature and volume of cosolvent seems to promote a greater homogeneity of the particles. By means of chromatographic analyses, the main compounds, responsible for the antioxidant activity, such as oleuropein, quercetin or apigenin among others were identified.

Keywords Rapid expansion · Supercritical · Olive leaves · Antioxidants · Quercetin · Apigenin

Introduction

A number of industries associated to the production of food additives, cosmetics or pharmaceutical products have increased their efforts to obtain bioactive compounds from agro-wastes by means of extractive and purification processes. The identification of new natural and safe resources as an alternative to obtain antioxidant substances especially from vegetal origin is on the focus of numerous

investigations. The fruit from Olea europaea L. var. euro- paea has been historical and widely used as food (they are part of the daily diet of a large part of the world’s popu- lation) and now olive leaves have attracted the interest of the scientific community and of many industries because of the numerous compounds of interest that they contain with regard to their beneficial effect on human health. Olive tree leaves are by-products resulting from the cultivation of olives, which is one of the most important crops in the Mediterranean region. Different research studies on olive leaves has revealed that their properties can be attributed to a group of secondary metabolites, such as flavones, flavonols, substituted phenols and also secoiridoids, such as oleacein or oleuropein [1–3].

The high content in these valuable compounds confers olive leaf extracts with potent antioxidant and anti-inflammatory properties.

According to different studies on the effect of olive leaf extracts on animals, they seem to have positive outcomes in

* A. Montes

[email protected]

1 Department of Chemical Engineering and Food Technology, Faculty of Sciences, University of Cadiz, International Excellence Agrifood Campus, (CeiA3)11510 Puerto Real (Cádiz), Spain

2 Departamento de Química y Ciencias Exactas, Universidad Técnica Particular de Loja, San Cayetano Alto sn, AP 1101608 Loja, Ecuador

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the treatment or prevention of several pathologies, such as cardiovascular disorders, hypertension, diabetes or hyper- lipidemia [4]. Moreover, olive leaf extracts have the ability to reduce blood pressure and to increase blood flow into the coronary arteries [5–7]. Olive leaf extract has also been proposed to be used as a food preservative [8].

Different methods have been used, optimized and compared for the extraction of the phenolic compounds in olive leaves. These go from conventional methods, such as solvent extraction or maceration, to advanced methods, such as ultrasound-assisted extraction (UAE) [9] or microwave- assisted extraction (MAE) [10]. Both UAE and MAE have been proven to obtain polyphenol-rich extracts with a higher antioxidant and antimicrobial activity in a shorter processing time and using less solvent [11, 12]. Olive leaf extracts have also been obtained through supercritical techniques using water, ethanol or a hydroalcoholic mixture (50:50) (v/v), but the greatest extract yield and with the highest antioxidant activity were achieved when ethanol was used as the pressurized solvent [13].

Many authors have investigated the phenolic compounds content in oil [14, 15] and leaves [16–18], because of their direct relation with their antioxidant activities. Of all the different parts in olive trees, leaves exhibit the highest antioxidant power [19]. Several groups of phenolic compounds can be found in olive leaves: oleuropeoside (oleuropein and verbascoside); flavones (luteolin-7-glucoside, apigenin- 7-glucoside, diosmetin-7-glucosise, luteolin and diosmetin);

flavonols (rutin and quercetin); flavan-3-ols (catechin);

and substituted phenols (tyrosol, hydroxytyrosol, vanillin, vanillic and caffeic acids) [3]. Oleuropein is the most abundant phenol in olive leaves, and its antiviral properties have already been demonstrated [20]. It can also be used to prevent heart diseases [21], to improve the metabolism of lipids [22] or to fight obesity. It also protects enzymes and possesses cytostatic and anti-angiogenic roles [23] among others. Some flavones, such as apigenin, present in olive leaves, have been associated to beneficial effects regarding Alzheimer’s [24], liver diseases [25, 26], atherosclerosis or restenosis [27]. It has been recently proven that this flavone could be used for the treatment of cervical cancer [28] or as an antiviral [29, 30]. On the other hand, some flavonols, such as quercetin have shown pharmacological activities such as antioxidant, anti-inflammatory, anticancer or antiviral [31].

The incorporation of these polyphenols into pharmacological formulations, cosmetic or food products comes associated to certain demands with regard to specific morphology, uniformity or particle size to achieve a better pen- etrability and dissolution rate and thus higher bioavailability (Fig. 1). Moreover, most of the processes associated to these industries preferably operate in solid state. Furthermore, many of the conventional methods that have been used until present to produce micro- and nanoparticles of the active substances present several drawbacks, such as their high processing temperatures, the use of large amounts of pol- luting organic solvents that may also damage the active

Fig. 1 Fish-bone of formulation processes

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substances of interest or leave residues in the final product and the difficulty to control particle sizes and uniformity [30, 32, 33]. In a previous work [34], supercritical antisolvent (SAS) was used to precipitate olive leaf antioxidant submicron particles. In this way, the initial extract, with a higher antioxidant activity associated to a higher concentration of oleuropein, was extracted by pressurized liquid [12]. The precipitated particles in these extracts presented large contents of polyphenols, such as oleuropein and tyrosol, with a higher antioxidant activity than the extract obtained in our study. Glycosides, a flavone family with a lower polarity and antioxidant activity, were not found in the extract, since they were probably washed away by the CO₂ and the solvent [12, 34].

Baldino et al. [35] used the same SAS technique at 35 °C and 150 bar to produce powder rich (36% w/w) in oleuropein microparticles from an olive leaf ethanolic solution with a high content of oleuropein. The particles with a higher oleuropein and a lower flavones content seemed to have a higher antioxidant activity, probably due to antagonist effects [13].

In this sense, most of the polyphenols used in extracts present synergic but also antagonist effects that affect their activities and properties. The purification of these extracts, at least by family of compounds, is an efficient strategy to increase the efficacy of these substances if intended to be used as a more specific treatment against certain disorders.

Rapid expansion of supercritical solutions (RESS) is considered to be a green process that uses supercritical carbon dioxide to generate micro- and nanoparticles. This technique gets round some of the drawbacks exhibited by other conventional methods, such as an excessive use of organic solvents, high processing temperatures or the need to use several purification steps, among others. What is more, by adjusting the density of the CO₂ the morphology and particle size of the precipitated can also be controlled.

On the other hand, Panagiotopoulou et al. [36] gave a one more step encapsulating a freeze-dried, enzymatically modified, aqueous extract of olive leaves using spray drying under optimized conditions in order to be used as cosmetic cream formulation and for food supplements. Microparticles of mean particle size around 14 µm were achieved whose main release was carried out in the small intestine. Moreo- ver, microparticles passed successfully the 6-month micro- biological test showing great stability.

RESS has been widely used to obtain valuable particles containing substances that are soluble in supercritical CO₂ [36–41]. However, there is not much literature on the use of RESS with complex matrices. In a previous work, orange leaves were processed by RESS to obtain an agglomerate without any defined particles [42]. On the other hand, RES- SAS (rapid expansion of supercritical solution into aqueous solutions), a modification of the RESS process where the supercritical solution is sprayed into an aqueous solution

instead of into the air, has been used to elaborate and stabi- lize potent antioxidant nanoparticles, such as those containing α-tocopherol or β-amyrin, from olives leaves [43].

This work intends to employ RESS to valorize olive leaves by extracting from them the main compounds of interest for the food, cosmetic or pharmaceutical industries.

In this process, a supercritical solution that contains other compounds that are soluble in CO₂ is sprayed through a nozzle into a precipitator. Since the solvent capacity of the CO₂ is dramatically reduced under atmospheric conditions, the solution supersaturates rapidly when the vessel is depres- surized and the fast nucleation that takes place results in the generation of nano or microparticles of the compounds that are found in olive leaves [44]. As expected from previous studies [13, 34], flavone and flavonol enriched precipitations in the nanometer range have been obtained from olive leaves using RESS. A secoiridoid as oleuropein was also precipitated. The effects from the main operating conditions of the RESS process on mean particle size, particle size distribution and antioxidant activity have been investigated.

Materials and methods

Materials

Ethanol absolute (99%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile and formic acid (HPLC grade) were supplied by Panreac (Barcelona, Spain). CO₂ with a maximum purity of 99.8% was obtained from Linde (Spain).

Olea europea leaves were collected in 2020 at Yunquera (Malaga, Spain). Leaves from four different varieties of olive trees were picked: hojiblanca, manzanilla aloreña, podrida and picual. The leaves were ground using a Bosch 6000 W grinder fitted with a 5 mm sieve and then they were oven dried at 45 °C and stored at room temperature in a well- ventilated shady place.

Experimental design

A Design of Experiment (DOE) was applied to clarify the influence from the main parameters on several variables responses, namely mean particle size, Polydispersity Index (PDI) and antioxidant activity of the particles obtained by RESS. The Statgraphics 19 Centurion application (The Plains, Virginia, USA) was used to analyze and screen the experimental conditions. A full factorial design (2⁴) with 16 factor points and 3 replicates at the center point was imple- mented. The total design consisted of 19 runs that were carried out at random.

Pressure (P) and temperature (T) inside the dissolving chamber, mass of olive leaves (M) and volume of cosolvent

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(C) were selected as the main parameter with an influence on the variable responses, and were, therefore, selected for the optimization of the RESS process to be applied to the olive leaf samples. The two levels established for each factor have been included in Table 1. Each factor top and bottom levels in the design range were selected according to previous experience. The nozzle diameter (100 µm), and contact time (60 min) were kept at constant values. The operating conditions used for the RESS process are shown in Table 2.

Rapid expansion of supercritical solution (RESS) The RESS equipment built by Thar Technologies® (Model RESS250, Pittsburgh, PA, USA) is shown in Fig. 2. The pilot plant includes a high-pressure pump for CO₂ (P1); a low-pressure heat exchanger to cool the CO₂ down to 5 ºC (HE1); an electric high-pressure heat exchanger (HE2) to heat the CO₂ up to the desired set point; a 250 mL stainless steel dissolving vessel (V1); an electrical heating jacket

wrapped around V1; a magnetic stirrer (maximum speed 2500 rpm); a stainless steel collection vessel (V2) where there particles precipitated once the supercritical solution was expanded through a 100 µm inner diameter stainless steel nozzle (Thar Technologies). This equipment has been described in further detail in a previous work [45].

The experiments were conducted according to the following procedure: first, the weighted ground leaves were placed inside the dissolving chamber together with the corresponding amount of ethanol. The system was held for 1 h (contact time) at 100 rpm in supercritical conditions to ensure its complete equilibrium. Then, the MV2 valve was opened for 60 s and the supercritical solution was sprayed through a nozzle, which has been pre-heated at 80 ºC in order to compensate heat losses and to prevent the nozzle from clogging during the rapid expansion of the solution.

The particles that precipitated from the olive leaves were collected for their subsequent analysis on the walls of vessel V2, which remained at atmospheric pressure.

Table 1 Two-level assessment for each factor and calculated effects on response variables

Factors Low level High level PS effects PDI effects AAI effects

Pressure (bar) 250 350 12.47 0.12 – 0.07

Temperature (K) 333 373 – 27.26 0.006 – 0.009

Mass of leaves (g) 2 4 119.44 – 0.009 0.006

Volume of cosolvent (mL) 9 18 – 22.35 0.05 0.009

Table 2 Experimental design and observed responses

P pressure, T temperature, M mass of olive leaves, C cosolvent, AAI antioxidant activity index, MPS mean particle size, PDI Polydispersity Index

Run name Run order P (bar) T(˚C) M (g) C (mL) Success AAI MPS (nm) PDI

1 8 250 60 2 18 + 0.29 ± 0.02 230 ± 150 0.39

2 4 350 60 2 9 + 0.12 ± 0.01 290 ± 120 0.15

3 2 350 100 2 9 + 0.13 ± 0.01 275 ± 100 0.12

4 6 300 80 3 13.5 + 0.57 ± 0.02 230 ± 100 0.18

5 5 250 60 4 9 + 0.14 ± 0.02 170 ± 10 0,002

6 1 350 100 4 9 + 0.23 ± 0.02 400 ± 270 0.55

7 9 300 80 3 13.5 + 0.54 ± 0.01 240 ± 100 0.19

8 11 250 100 2 18 + 0,17 ± 0.06 110 ± 55 0.23

9 13 300 80 3 13.5 + 0,49 ± 0.03 230 ± 110 0.22

10 18 250 100 2 9 –

11 3 250 100 4 18 + – Agglomerated –

12 14 350 60 2 18 + 0.14 ± 0.01 110 ± 60 0.32

13 7 250 60 2 9 –

14 10 350 60 4 18 + 0.17 ± 0.01 150 ± 70 0.20

15 17 250 60 4 18 + – Agglomerated –

16 16 350 60 4 9 –

17 15 350 100 4 18 + 0.15 ± 0.01 60 ± 30 0.18

18 12 350 100 2 18 + 0.18 ± 0.01 55 ± 20 0.15

19 19 250 100 4 9 – 230 ± 150

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Sample characterization

A Nova NanoSEMTM 450 Scanning Electron Microscope (SEM) was used to evaluate the morphology and size of the olive leaf precipitated samples. The precipitated samples were covered with a 15 nm gold coating by means of a sput- ter. The SEM images were processed using the Scion image analysis application (Scion Corporation) to determine the particle sizes. Approximately, 300 particles were analyzed in each experiment. The mean particle size and particle size distribution were determined by means of the Statgraphics Centurion XVI application.

The separation and identification of the different olive leaf compounds was conducted by ultra-performance liquid chromatography (UHPLC) coupled to a quadrupole-time- of-flight mass spectrometry (QToF-MS) (Xevo G2 QToF, Waters Corp., Milford, MA, USA). A UPLC HSS T3 column of 1.7 μm particle size (2.1 × 100 mm) manufactured by Waters (Milford, MA, USA) was used. The mobile phase was a binary solvent system consisting of water with 0.1%

formic acid (phase A) and an acetonitrile with 0.1% formic acid (phase B). The column flow and temperature were set at 0.6 mL/min and 40 °C, respectively. A gradient elution program was used with both phases: the elution was started at 90% phase A for 0.5 min; then, it was changed to 0% A after 3 min and remained at 0% for 0.7 min. After 0.6 min,

%A was increased to 90% and kept constant for 0.2 min.

The electrospray was operated in negative ionization mode and for full scan analysis (100–700 Da), using a capillary voltage of 3 kV, a cone voltage of 30 V, a cone gas flow of 10 L/h, 120 °C as source temperature, 450 °C desolvation temperature and 800 L/h desolvation gas flow, as ion source parameters. The collision energy ramp for MS was set at 6 eV and the scan time for the lock mass was set at 0.2 s with 3 scans to calculate their average and a mass win- dow of ± 0.5 Da. Previously to their analysis, the samples were filtered through a 0.2 μm nylon syringe filter and the

injection volume was set at 1 µL. The calibration curves for ten standards (Table 3) that were previously elaborated [46]

have been used to identify main compounds of precipitates.

All the analyses were performed in triplicate and the standard deviation was calculated in each case.

Antioxidant activity

The ability of the particles obtained by RESS from olive leaves to scavenge DPPH free radicals was determined according to the method described by Scherer and Godoy [47]. The aliquots (90 µL) of six samples at different con- centrations were mixed with 3510 µL of a DPPH working solution (6 × 10^–5 mol DPPH/L ethanol). These solutions were mixed and then stored in the absence of light for 3 h to allow the reaction to take place. The final absorb- ance was measured at 517 nm. For the quantification of the antioxidant capacity, a standard curve for DPPH was

Fig. 2 RESS250 lab scale unit; CO₂ pump (P1); solubilization vessel (V1); collection vessel (V2); low-pressure heat exchanger (HE1); electric high-pressure heat exchanger (HE2); manual valves (MV1 and MV2)

Table 3 The analytical characteristic for determination of olive leaves compounds (data collected from [46])

rt retention time, M-H⁻ molecular ion; ^*sample size = 7

Compounds rt [M-H⁻] Linear equation^* i² Hydroxytyrosol 1.20 153.0552 y = 2630.91x + 0 0.999 Caffeic acid 1.86 179.0344 y = 10959.1x + 0 0.994 Rutin hydrate 2.05 609.1456 y = 7511.57x + 0 0.993 Verbascoside 2.14 623.1976 y = 6729.6x + 0 0.999 Luteolin-7-glu-

coside 2.18 447.0915 y = 5906x + 1947.72 0.999 Apigenin-7-glu-

coside 2.29 431.0978 y = 7141.36x + 0 0.996 Oleuropein 2.38 539.1848 y = 12987.6x + 0 0.990 Luteolin 2.62 285.046 y = 15068.7x + 0 0.993 Quercetin 2.66 301.0344 y = 9108x + 0 0.997 Apigenin 2.79 269.045 y = 18923.8x + 23790.8 0.999

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prepared at different concentration levels between 7 × 10^–5 and 6 × 10^–6 mol/L. All the calculations were performed in triplicate. The results are expressed in μg/mL.

The percentage of DPPH remaining was calculated using the following equation, where C_DPPHt is the final DPPH concentration (3-h reaction, previously determined) and C_DPPHo is the initial DPPH concentration (0-h reaction):

The IC₅₀ value was graphically calculated using a poly- nomial fitting curve by plotting the sample concentration vs.

the % DPPH remaining.

The antioxidant activity was expressed as the antioxidant activity index (AAI) according to the following equation:

Results and discussion

Analyses of the design of experiments

The results from the ANOVA of the model with regard to mean particle size and their antioxidant activity are sum- marized in Table 4 and include the degree of freedom (DF), sum of squares (SS), mean square (MS), coefficient of determination R2 (adjusted and experimental) and goodness of prediction (Q2). Based on F and p values, we can conclude that the model is significant for mean particle size diameter and no significant for antioxidant activity and PDI [48].

The mean particle size p-value was lower than 0.05, while a higher F value indicates that this variable is significant, as can be seen in Table 4. In this case, the probability of lack of fit is not significant at 95% and, therefore, it can be concluded that the model presents no statistical lack of fit. Consequently, the model should allow to determine the optimum operating conditions that would yield particles of a smaller mean size from olive leaves. On the other hand, (1)

%DPPH remaining = C_DPPHt C_DPPHo × 100

(2) AAI =

C_DPPHt IC50

since the model is not significant for PDI, it cannot be used to obtain a narrower and optimized size distribution.

Figure 3 and Table 1 present, in decreasing order, the values of the factors and their interactions with any influence on the mean particle size. Pressure x volume of cosolvent exhibited the greatest influence on the mean particle size, followed by the mass of leaves. Pressure x mass of leaves and temperature x volume of cosolvent had moderate effects on the mean particle size. However, neither pressure nor temperature as separate factors had any significant effect on the mean particle size. On the other hand, none of the statistical parameters was analyzed for antioxidant activity index or PDI since the model was not adjusted and, therefore, these values were not significant.

RESS process

The RESS process led to a powder precipitation in most of the assayed experimental conditions thanks to the solubility in supercritical CO₂ of part of the compounds that are found in olive leaves. Spherical submicron particles were obtained as can be seen in Fig. 3. The smallest nanoparticles (run 18, Fig. 4) were achieved when using higher pressure and temperature and a greater amount of cosolvent. However, in some cases, the precipitation failed to occur (runs 10, 13, 16 and 19 in Table 2) or larger particles with irregular shapes (runs 11 and 15 in Table 2) were obtained. The failing experiments were those with lower amounts of cosolvent, which

Table 4 Analysis of variance for design model of process variables

Degrees of Freedom (DF), Sum of Squares (SS), Mean Square (MS)

Variables DF SS MS R² AdjR² Q² Significance

F Lack of fit

p

MPS Model 13 118,019 9078.40 0.91 0.63 0.20 4298.45 0.004

Residual 3 10,034.4 3344.80

PDI Model 13 0.32 0.02 0.97 0.89 0.36 240.49 1.13

Residual 3 0.01 0.03

AAI Model 13 0.34 0.03 0.10 – 2.90 0.00 18.91 1.00

Residual 3 0.30 0.10

Fig. 3 Effect of the considered variables on mean particle size of precipitated particles from olive leaves

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could indicate that more cosolvent is required to change the polarity of CO₂ and cause the precipitation of the olive leaf supercritical solution. However, some of the other experiments where the same amount of cosolvent had been used, succeeded to achieve the precipitation of the particles, which may indicate a complex interaction between certain variables. To further investigate this aspect, a possible correla- tion between mean particle size and experimental parameters was examined.

For that purpose, the effects of the experimental parameters on the mean particle size are plotted in Fig. 5. It can be observed from this graph that lower pressures generally led to smaller mean particle size. On the other hand, both higher temperature levels or greater volumes of cosolvent would result in smaller particles. This fact can be clearly observed from the data corresponding to experiments 1, 17 and 18.

Thus, runs 17 and 18 produced the smallest particles from olive leaves, while, although run 1 also produced submicron particles, they were of a larger size, since they were precipitated with the low volume of cosolvent. With regard to

the mass of olives leaves, it exhibits a direct effect on mean particle size, so that smaller particles were obtained when smaller amounts of olives leaves were used. Thus, more than

Fig. 4 SEM images of olives leaves particles precipitated by RESS process

Mean Particle Size (nm)

Pressure

TemperatureMass of leaves

Volume of Cosolvent 140

170 200 230 260 290

Fig. 5 Main effect of process parameters upon mean particle size of olive leaves particles

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2 g of leaves did not increase the saturation of the supercritical solution nor resulted in smaller particles (Fig. 5).

In any case, the results that could be observed from the matrix design indicated an opposite effect in certain cases, which was probably due to the effect of the interactions between some of the variables.

Consequently, the plots representing the interactions between all the factors were examined (Fig. 6). In this figure, low level (–) and high level (+) of the variable A have been represented for low (left side) and high (right side) levels of variable B. No relevant variations of the mean particle size associated to the effect of the interaction between pressure and temperature could be observed.

Thus, a reduction of the mean particle size was obtained when the temperature was increased, regardless of the pressure variation within the range considered in the experiments. In addition, the solvent power of CO₂ does not seem to be relevant regarding the precipitation of olive leaf compounds within the assayed levels. There was some interaction between mass of leaves and volume of cosolvent, so that as the volume of the solvent was increased, the particles were smaller and this trend was more pronounced when the volume of leaves was lower. Therefore, particles were bigger when the volume of leaves was greater and the volume of the solvent decreased. On the other hand, the effect from the interaction between pressure and volume of cosolvent was more significant. Thus, at lower pressure levels, smaller mean particle size was achieved while the volume of cosolvent was also low. However, when pressure was high, smaller particles were achieved when the volume of cosolvent was increased. In this sense, pressure is in line with volume of cosolvent due to when volume of cosolvent is increased higher pressure and amount of CO₂ would be necessary to solubilize the solvent and form a saturated supercritical solution with the olive leaves. The effect from

the interaction of temperature and volume of cosolvent was also rather noticeable, so that at a low temperature, as cosolvent volume was increased, larger particles were obtained, while at high temperatures, as the volume of cosolvent was increased, smaller particles were obtained.

Particle size uniformity has been determined as a func- tion of Polydispersity Index (PDI) (Fig. 7), since it could be observed that the mean particle size was directly related to PDI. Thus, the largest particles (like those from run 6) had the highest PDI, which indicates larger size particles and more uneven particle sizes (Table 2). The runs 17 or 18 produced smaller particles, which corresponded with a low PDI and more even particle sizes. The lowest PDI corresponded to the particles obtained from run 5.

Antioxidant activity

The antioxidant activity indexes of the precipitates are presented in Table 2. In general, the AAI (antioxidant activity index) of most of the RESS precipitates is low (AAI < 0.5) and only those corresponding to the central points could be considered as moderate (0.5 < AAI < 1) according to the scale established by Scherer and Godoy [45]. In this way, it can be observed in Table 2, that the highest AAIs correspond to the precipitates from runs 1, 4, 7 and 9. Since, runs 4, 7 and 9 belong to the intermediate values in the design, these operating conditions are the ones recommended to obtain compounds with a higher antioxidant activity. On the con- trary, runs 12, 14 and 17, which were conducted at higher pressure levels (350 bar) presented lower AAIs. Given that the model was not significant for AAI, no clear trend could be associated to the range of operating conditions that had been studied. In any case, a high-pressure level does not seem to be advisable when intending to obtain high antioxidant compounds.

Let us mention a study by Chinnarasu et al. [34] where olive leaf extracts obtained through supercritical CO₂ was employed to precipitate particles by supercritical antisolvent (SAS) under varying operating conditions. The nano- particle precipitates obtained by these authors presented an AAI of 1.35, which meant that the AAI of the initial extract (0.32) had been multiplied by nearly five. This considerable enhancement of the particles’ AAI was mostly attributed to the polar compounds with antioxidant properties, such as polyphenols, which did not dissolve in the CO₂ and, instead, got precipitated through the antisolvent process. In the case of precipitates obtained through RESS, their AAIs are simi- lar to those exhibited by olive extracts, but lower than those obtained through SAS. This would be explained by the fact that the precipitates obtained through RESS would mostly contain non-polar compounds, while hardly any polar compounds would be part of them.

Mean Particle Size (nm)

AB

- -

+ +

AC -

- +

+

AD -

- +

+ BC -

-

+ +

BD - + -

+

CD -

-

+ +

90 130 170 210 250 290 330

Fig. 6 Interaction plots of parameters on the mean particle size of olive leaves particles (A pressure; B temperature; C mass of leaves; D volume of cosolvent)

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Nikos Xynos et al. [49] also measured the AAI of the olive leaf extract obtained by supercritical extraction and pressurized liquid extraction (PLE). The serial combination of supercritical CO₂ modified by ethanol at 5% and subcritical water resulted in large extraction yields (44.1%), a high recovery of oleuropein (4.6%) and a notable AAI (145,3 µ/

mL DPPH). First, supercritical CO₂ was used to extract non- polar and lipophilic non-desirable (fats, waxes, chlorophyll) compounds. Then, by PLE the interesting polar compounds were isolated with the aid of the necessary amount of ethanol as cosolvent to extract oleuropein. In that procedure, the high yields obtained were attributable to the high percent- ages of ethanol being used [50].

UPLC‑MS analysis

There is no clear relation between the main parameter of the RESS process and the antioxidant activity of the precipitates.

However, the differences between the AAI of the precipitates from runs 4 and 9 and the AAI of those from the runs 14 and 18, with higher AAI exhibited by the former, could be due

Fig. 7 Particle size distribution of olives leaves particles precipitated by RESS process

Fig. 8 UPLC chromatograms of precipitates

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to the different composition of the precipitates. Chromato- graphic analyses of representative samples (runs 1, 2, 4, 6, 7, 9, 14 and 18) were carried out in order to identify those differences in composition that could have an influence on their antioxidant properties (Fig. 8).

Ten standards, luteolin 7-glucoside, apigenin, quercetin, rutin hydrate, apigenin 7-glucoside, oleuropein, verbascoside, hydroxytyrosol, caffeic acid and luteolin were measured by UPLC-MS in order to identify these compounds in the precipitates [46] (Table 3). In general, RESS precipitates contained mainly between one and five of the compound that have been previously mentioned. The results have been collected in Table 5, even if they could not be quantified because of their low concentration levels below those of the standard used to generate the curve.

The chromatograms of the runs 4, 7 and 9 correspond to the intermediate values where a higher number of polyphenols, such as luteolin-7-glucoside, oleuropein, luteolin, quercetin and apigenin had been identified. These precipitates also presented the highest antioxidant activity. The precipitate from run 1 was composed by luteolin-7-glucoside, oleuropein and luteolin. The one from run 6 was composed by quercetin, apigenin and apigenin-7-glucoside.

Both experiments (1 and 6) produced precipitates with lower antioxidant activity than those from runs 4, 7 and 9, probably due to their lower polyphenol content. The precipitate from run 18 only contained quercetin and apigenin and the precipitates from runs 2 and 14 were composed only by apigenin.

This low number of polyphenolic compounds is related with the poor antioxidant activity of these precipitates. However, in these runs 2 and 14, the more selective precipitation was achieved.

According to the UPLC-MS analyses, RESS, and mostly when applied at intermediate level parameters, seem to produce precipitates from which a number of nanoparticles formed by compounds with antioxidant activity such as quercetin, oleuropein, luteolin or apigenin can be obtained in an environmentally friendly manner.

Conclusion

Supercritical CO₂ has been used to extract and precipitate, through a one-step process, antioxidant compounds from olive leaves, a waste generated during the harvest- ing of olives to produce olive oil. RESS was applied to olive leaves to produce spherical particles in the nano- or submicron range. The mean size of the particles could be reduced by decreasing the pressure or increasing the temperature in the dissolving chamber. It seems that the amount of cosolvent should be in line with the level of pressure to reduce the mean particle size. Using a limited mass of leaves was also an effective method to precipitate smaller particles. This means that there are different ways to adjust the mean size of the final particles by varying different operating conditions. Moreover, lower pressure, temperature and volume of cosolvent seems to be adequate to get lower Polydispersity Index and thus higher homogeneity.

The antioxidant activity index of the precipitates was determined between 0.63 and 0.14, which could be explained according to the presence of polar compounds in the precipitates. Only by increasing the pressure up to 250 bar AAI could be increased. In any case, a number of particles of interest could be obtained after purifying the precipitates, such as quercetin, oleuropein, luteolin or apigenin. RESS when applied at the mild conditions studied has proven to be an effective method to obtain from olive tree leaves nano- and microparticles formed by compounds with interesting antioxidant properties and that could be of practical use in the food, pharmaceutical or cosmetic industries.

Acknowledgements We gratefully acknowledge the Spanish Minis- try of Economy, Industry and Competitiveness (Project CTQ2017- 86661-R) and European Regional Development Fund (ERDF) for financial support, and Central Service of Science and Technology of University of Cadiz for analyses.

Table 5 Identified compounds by UPLC-MS in olive leaves’

precipitates

Compounds Luteolin-

7-glucoside Oleuropein Luteolin Quercetin Apigenin Apigenin- 7-glucoside

Run 1 + + + – – –

Run 2 – – – – + –

Run 4 + + + + + –

Run 6 ^a – – – + + +

Run 7 + + + + + –

Run 9 – + + + + –

Run 14 – – – – + –

Run 18 – – – + + –

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Declarations

Conflict of interest The authors declare that they have not conflict of interest.

Compliance with ethic requirements This article does not contain any studies with human or animal subjects.

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